Production and Nutritional Quality of Tomatoes (Solanum lycopersicum var. Cerasiforme) Are Improved in the Presence of Biochar and Inoculation with Arbuscular Mycorrhizae

Tomato is a fruit of great nutritional interest in 
the basic human diet. The increasing use of agrochemicals to maintain 
production requires new alternatives to reduce environmental impact. Arbuscular mycorrhizae (AM) 
are beneficial microorganisms that favor the growth of plants improving their 
nutrition and development, protecting the plant from biotic and abiotic 
stresses and favoring the production of bioactive compounds that increase their 
nutritional value. The use of biochar as soil conditioner is also considered an 
environmentally friendly resource. A greenhouse experiment was carried out to 
observe the effect of the use of biochar and AM inoculation on the quality of 
fruits, yield and polyphenols production of Cherry tomato, Solanum lycopersicum var. Cerasiforme. A mixture of rice husk 
biochar with sterile sand and two inoculums of Glomeromycota native fungi: from 
a wetland (GWI) and a fallow field (GFI) were used. Control treatments 
consisted of inoculation with both GWI and GFI in sterile sand. All treatments 
were irrigated with 50% La Molina? hydroponic solution. After 12 weeks plants 
were harvested to quantify weight, number and diameters of the fruits, and 
yield, total 
polyphenols in the fruit pulp were quantified. In the presence of biochar and 
the two inoculums, GFI and GWI, fruit production was favored throughout the 
experiment. The height of the plants was significantly greater in the presence 
of biochar. Plants grown in biochar and inoculated with GFI had a yield of 8.2 
MT/Ha, increasing in 50% this value respect to control with biochar (5.33 
MT/Ha). This treatment doubled the number of fruits (59.5) with respect to the 
control (32.5). Root colonization by GFI was not affected by the presence of 
biochar. It is concluded that the combined use of rice husk biochar and Glomeromycota 
fungal inoculation is recommended for increasing of Cherry tomato yield and 
improving fruit quality through the production of bioactive compounds.


Introduction
Tomato is a vegetable of nutritional interest and high consumption in the daily diet. In Peru, about 13,796 tons were produced in 2018, in an area of 6000 ha (2.29 ton/ha), a production that was mostly intended for internal consumption [1]. The need to maintain and increase production has led farmers to use increasingly high doses of chemical fertilizers with more frequent applications. The recurrent use of these products must be controlled with alternatives that reduce their impact on the soil, crops and environmental effects. Beneficial microorganisms constitute an option, including growth promoting bacteria, of which there are successful applications in Peru, favoring yield thanks to their ability to promote the plant development [2]. Arbuscular mycorrhizae (AM) are also considered as one of the organisms of importance in agriculture due to their ability to promote nutrition and plant growth by capturing nutrients, mainly P, thanks to the mycelium of Glomeromycota fungi distributed externally to the roots of the plants they colonize symbiotically [3]. Arbuscular mycorrhizae have also proven to be effective in favoring the tolerance to biotic and abiotic stresses of plants and soil structure; they also behave as agents which improve the quality of fruits and/or foods, encouraging the production of bioactive compounds [4] with antioxidant capacities able to enhance their nutritional value [5].
In the agriculture scenario, the ecological impact of fertilization and constant sowing on the soil, degrading it and reducing its fertility must be mitigated. The use of biochar, a carbon-rich substrate produced by pyrolysis [6] from plant biomass used as amendment to induce changes on soil properties, fertility and micro-organisms abundance and/or functionality, including mycorrhizal fungi [7], has become an alternative for sustainable agricultural management. When applied to the soil, it resists decomposition, effectively sequestering the applied carbon and mitigating anthropogenic CO 2 emissions [8], so it is highly recommended as a soil restoring agent [9] [10]. Likewise, several authors refer to the suitability of biochar as support for biofertilizers [11], being this characteristic an added value to the present experimentation. In order to study tomatoes cultivation alternatives promoting an increase in its yield through an environmentally friendly management, an experiment was carried out to test production by evaluating biochar as part of the substrate together with arbuscular mycorrhizae, its effect on tomatoes yield and on the quality of bioactive tomatoes compounds.
Biochar particles were sieved and those retained between 500 and 2000 µm sieves were selected. 4 kg pots containing a mixture of rice husk biochar and sand in a 1:3 ratio were used. The mixture was sterilized with fluent steam (autoclave) for 1 hour every 24 hours for three days [12].

Greenhouse Experiment
The experiment was installed in the greenhouse of the Laboratory of Microbial Ecology and Biotechnology at La Molina National Agrarian University. Experimental conditions were: 12 h photoperiod, maximum and minimum temperature (32˚C and 17˚C) and 89% of relative humidity. The experiment was carried out between January and April 2019, lasted 12 weeks.
Biological material. Cherry tomato seeds, Solanum lycopersicum var. Cerasiform, were disinfected with 3% sodium hypochlorite (15 minutes) and then rinsed with sterile water. Seeds were placed to germinate in sterile peat trays,  [13] In each case, the spores were multiplied in two cycles of 4 months each; rhizospheric samples were taken from the second multiplication, Glomeromycota spores [14] and % root AM colonization [15] were quantified. A similar number of spores of GFI and GWI inoculums were procured to inoculate in each mycorrhized treatment of the experiment. GFI inoculum was formed by the native species mainly by the fungus R. intraradices and the genera Acaulospora, Gi- Quantification of arbuscular mycorrhizae root colonization. From the rhizospheric soil of each mycorrhized plant (treatments T2, T3, T5 and T6) 2 cores (1 inch diameter) were extracted, mixed and a subsample of roots was extracted to clear and stain with trypan blue [16] and quantify mycorrhizal colonization [15]. These determinations were performed in quadruplicate in each treatment.
Determination of polyphenols in the fruit. The methodology according to [17] was applied with modifications for preparing the sample. Collected fruits were washed with distilled water to remove dirt and impurities, and disinfected with alcohol. The fruits of all repetitions per treatment were taken, mixed and 4 ripe fruits without mechanical damage were chosen and ground in mortar with liquid N. The compound obtained was filtered and 25 mL of this liquid was mixed with 25 mL of methanol (analytical grade). The mixture was centrifuged at 350 rpm for 10 minutes. The supernatant was separated in a graduated tube and the volume adjusted to 10 mL with cold methanol. 500 µL of the test sample and 250 µL of the Folin-Ciocalteu reagent were taken; 1.25 mL of 7.5% sodium carbonate w/v were added, the mixture was stirred and left for one hour in darkness. After one hour the absorbance was read on a Thermo spectrophotometer, Model Genesis 6 at 755 nm. The value of total polyphenols is expressed as equivalents of Gallic Acid/ 100 g fresh weight (mg GAE/100 g FW).
Brix grade determination. 1 drop was taken from the filtered juice of fruits obtained above and read on a PAL-1 refractometer (ATAGO), the results are expressed in % soluble solids or ˚Brix.
Statistical analysis. For all measured variables, the analysis of variance was performed with a 95% of confidence using the F test to determine the difference between treatments. Duncan test was applied for means comparisons between treatments, using the statistical software SPSS version 24.

Results and Discussion
Fruit production. The formation of fruits from week 5 to week 12 (Table 1) was evaluated, observing an increase in fruits over time. Treatments with biochar, GFI and GWI inoculums showed the greatest significant number of fruits W. Castañeda et al. compared to the control of plants grown only in sand (T1) and with biochar (T4). Additionally, treatment with GWI inoculum also presented a higher number of fruits with significant differences with respect to the control without mycorrhiza (T4).
Plant response. Table 2 shows that plants grown with biochar showed significantly greater height than those grown without it. Biochar is known for its ability to improve growth and production of crops (Rawat et al. 2019).
It is generally recognized that biochar benefits plant nutrition [9] and its development [8]. Since biochar is derived from plant biomass, it is high in carbon and may contain a range of nutrients suitable for the plant, which may improve yield as shown in Table 2. Some elements that are still in very low proportions can be used by the plant as for example nitrogen, potassium or iron among others. Also, the high moisture retention capacity of biochar could help reducing water loss from the substrate. Physical microstructure may crucially influence the role of biochar on plant nutrient uptake influencing access to mineralized elements by soil solution, microorganisms, and plant roots [18]. The beneficial use of biochar as soil amendment in terms of increased crop yield and improved soil quality has been reported and agrees with our results.
Such beneficial effects are also reflected in the yield obtained by applying biochar and more sharply together with the mycorrhizal inocula (treatments T5 and T6) (  In the case of GWI inoculum, AM colonization was similar with and without biochar, however tomato plants in the presence of biochar inoculated with GFI showed the highest yield recorded in the experiment, 54% over the control (T4).
The dissimilarity of the colonizing effectiveness of the inoculums may be due to different species of fungi present in each of them, as [21] observed when using consortium inoculums of several Glomeromycota species. The different ability of fungi to take nutrients in the substrate, the interaction of fungi with biochar, the distinct amount of mycelium produced by the fungi of each inoculum reflected in unequal efficiency of taking P [22], along with the dissimilar affinity shown by Glomeromycota species in each inoculum with tomato plants [23], might have influenced the responses observed in our experiment. Chlorophyll content. Treatment with biochar and GWI (T6) showed the highest chlorophyll content with respect to all treatments, even higher than the content level shown at its control without mycorrhizae (T4). In general, it has been observed that mycorrhizal inoculation improves chlorophyll content of the plant in crops of edible interest [24] [25] and photosynthetic activity [26]. However, chlorophyll content in mycorrhized plants and their fruits may possibly decrease due to the ripening process that favors the degradation of chlorophyll and accumulation of other pigments in the fruits [27] and leaves, effect that may be appreciated with GFI inoculum (T5). The higher content of chlorophyll in the presence of biochar and GWI (T6) could be related to the higher content of chloroplasts in the leaves of inoculated plants, as [28] found in mycorrhized plants.
Fruit number and quality. Regarding fruit number (Table 1 and Table 3), the inoculation with GFI and biochar stands out, obtaining around 50% more fruits than the respective control without mycorrhizae (T4). Likewise, the application of both inoculums was effective in the absence of biochar (T2 and T3), Table 3. Diameter, weight, brix grades and total polyphenol content (mg GAE/100 g FW) of the fruits of tomato plants S. lycopersicum var. Cerasiform, grown in the presence or absence of rice husk biochar and inoculated with two Glomeromycota fungi inocula. with up to 45% more fruit content than the control (T1). In relation to fruit characteristics (Table 3) Similarly, the higher content of total polyphenols of the fruits with inoculation of GFI and biochar is remarkable, suggesting that both factors favor the formation of bioactive compounds such as polyphenols in fruits. Our results coincide with [32] who demonstrated that inoculation with AMF enhances both phenolics and antioxidant activity, suggesting a positive influence of AMF inoculation on yield and on nutraceuticals production. Some mechanisms are suggested to explain the accumulation of secondary metabolism compounds in the plant in American Journal of Plant Sciences response to mycorrhizal symbiosis as an improvement in the nutritional condition of the host, such as alterations in the activity of key-enzymes, activation of metabolic routes, and increase in gene expression [33], some of which may be happening in our case.
However, this result coincides with the highest value of polyphenols recorded for the absolute control treatment (T1), which may be related to the nutritional stress achieved by the plant grown in nutritional deficit conditions (only with 50% nutrient solution), lacking mycorrhizal inoculation and/or developing in a low nutrient substrate [34] [35], influencing plant response with the highest production of antioxidant compounds [36].
The high values of total polyphenols recorded in this work-in the order of 28 mg GAE/100 g fresh weight-are higher than those registered for other tomato varieties different from the variety used in this work, both with and without biochar. [37] recorded 10 mg GAE/100 mg for various tomato varieties or 12 mg GAE/100 mg [38], which illustrates the effectiveness of the use of biochar and mycorrhizae arbuscular nutrition of Cherry tomato (T6). Our data should be compared with other measurements of polyphenols and bioactive substances in Cherry tomato, at present lacking in scientific literature.
Over the past several decades, many researches have pointed out the direct relation between the intake of bioactive compounds present in tomato and a reduced risk of suffering different types of cancer. These bioactive constituents comprise phytochemicals such as carotenoids and polyphenols. The ability of phenolic compounds as reactive oxygen scavengers is the main motivator of its antioxidant properties [38], which highlights the importance of T6 treatment in stimulating the production of polyphenols and the greatest yield of tomato.

Conclusions
According to our results, plant height, chlorophyll, fruit number, yield and polyphenol production were improved in tomatoes when inoculated with Glomeromycota fungi and grown with rice husk biochar. Mycorrhizal symbiosis and biochar elicited changes in the levels of total polyphenols. The significant polyphenol production and yield shown by tomato plants inoculated with GFI and biochar points out not only a greater nutritional and antioxidant value but also a defense role against herbivores and phytopathogens. The relationships between AM fungal inoculations, biochar addition and polyphenol production in tomato and other plants is an understudied area. Our findings can be used to improve this understanding as well as the potential of using biochar and AM.